Search

Che-Hsing Li,1,2,&ast; Jiunn-Liang Ko,1,3,4,&ast; Yu-Ping Hsiao,5,6 Ming-Hung Tsai,7 Yen-Chein Lai,8 I-Lun Hsin,3,4 Yu-Ting Kang,3,4 Gwo-Tarng Sheu,1,3,4 Wea-Lung Lin,6,9 Ming-Fang Wu1,4,6 1Divisions of Medical Oncology and Pulmonary Medicine, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, 402, Taiwan; 2Graduate Program in Immunology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA; 3Institute of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan; 4CSMU Lung Cancer Research Center, Chung Shan Medical University, Taichung, 402, Taiwan; 5Division of Dermatology, Chung Shan Medical University Hospital, Taichung, 402, Taiwan; 6School of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan; 7Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung, 40447, Taiwan; 8Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, 402, Taiwan; 9Department of Pathology, Chung Shan Medical University Hospital, Taichung, 402, Taiwan&ast;These authors contributed equally to this workCorrespondence: Ming-Fang WuDivisions of Medical Oncology and Pulmonary Medicine, Department of Internal Medicine, Chung Shan Medical University Hospital, 110, Sec. 1, Jianguo N. Road, Taichung, 40201, TaiwanTel +886-4-24739595#34711Email mfwu0111@gmail.comPurpose: Pemetrexed-based chemotherapy (Pem-C) is the first-line chemotherapy for advanced non-squamous non-small cell lung cancer (NSCLC). However, limited tumor-associated proteins in blood are available to predict pemetrexed response and/or survival.Patients and Methods: Plasma samples from three responders and three nonresponders with stage IIIB–IV NSCLC were collected prior to Pem-C and analyzed using Proteome ProfilerTM Human XL Oncology Array to detect 84 oncology-related proteins. The plasma concentrations of cathepsin S, endoglin (ENG), and matrix metalloproteinases 3 and 9 in 71 patients with advanced NSCLC treated with Pem-C were further measured using enzyme-linked immunosorbent assay based on the remarkable differences in the four proteins between responders and nonresponders in the array results.Results: Pem-C responders had significantly higher ENG levels but not the other three markers than nonresponders (mean ENG level: 27.1 ± 7.4 vs 22.3 ± 6.9, p < 0.01). High ENG concentration was correlated with improved progression-free survival (hazard ratio [HR]: 0.52, 95% confidence interval [CI]: 0.31– 0.86, p < 0.01) and overall survival (HR: 0.55, 95% CI: 0.32– 0.94, p < 0.05) in patients treated with Pem-C, and the ENG level was an independent factor in our cohort (HR: 0.54, 95% CI: 0.33– 0.89, p < 0.05). ENG concentration in Pem-C responders also significantly increased at the time of best response (p < 0.05).Conclusion: Cumulatively, this study reveals that ENG is correlated with Pem-C responsiveness in patients, which indicates the potential use of plasma ENG levels as a non-invasive biomarker for pemetrexed-based treatment in patients with non-squamous NSCLC.Keywords: endoglin, pemetrexed-based therapy, prognostic factor, non-squamous non-small cell lung cancer, biomarker

Integrins, the receptors of the extracellular matrix, are critical in the proliferation and metastasis of cancer cells. GMI, a Ganoderma microsporum immunomodulatory protein, possesses anticancer and antivirus abilities. The object of this study is to investigate the role of GMI in the integrins signaling pathway in lung cancer cells that harbor the EGFR L858R/T790M double mutation and osimertinib-resistance. Liquid chromatography-mass spectrometry and western blot assay were used to investigate the effect of GMI on inhibiting the protein expressions of integrins in H1975 cells. The migration ability and xenograft tumor growth of H1975 were suppressed by GMI. To elucidate the role of the integrin family in lung cancer resistant to osimertinib (AZD-9291, Tagrisso), H1975 cells were used to establish the osimertinib-resistant cells, named H1975/TR cells. The expressions of Integrin αV and stemness markers were much higher in H1975/TR cells than in H1975 cells. GMI suppressed cell viability, tumor spheroid growth, and the expressions of integrin αV and β1 in H1975/TR cells. Furthermore, GMI suppressed the expressions of stemness markers and formation of tumor spheres via blocking integrin αV signaling cascade. This is the first study to reveal the novel function of GMI in constraining cancer stem cells and migration by abolishing the integrin αV-related signaling pathway in EGFR-mutated and osimertinib-resistant lung cancer cells., (© 2024 Wiley Periodicals LLC.)

The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of β-catenin are commonly detected in endometrioid endometrial cancer. ICG-001 is a β-catenin inhibitor via blocking the complex formation of β-catenin and cAMP response element-binding protein (CREB)-binding protein (CBP). This study aims to investigate the effect of ICG-001 on endometrial cancer inhibition. First, endometrial carcinoma patient-derived xenograft (PDX)-derived organoids and primary cells were used to verify the inhibiting ability of ICG-001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG-001. Using MTT assay and tumor spheroid formation assay, ICG-001 significantly reduced the cell viability of HEC-59 and HEC-1A cells. ICG-001 enhanced cisplatin-mediated cytotoxicity. ICG-001 decreased cancer stem cell sphere formation. ICG-001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG-001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG-001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG-001-mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG-001 against endometrial cancer., (© 2023 Wiley Periodicals LLC.)

Cachexia is a lethal muscle-wasting syndrome associated with cancer and chemotherapy use. Mounting evidence suggests a correlation between cachexia and intestinal microbiota, but there is presently no effective treatment for cachexia. Whether the Ganoderma lucidum polysaccharide Liz-H exerts protective effects on cachexia and gut microbiota dysbiosis induced by the combination cisplatin plus docetaxel (cisplatin + docetaxel) was investigated. C57BL/6J mice were intraperitoneally injected with cisplatin + docetaxel, with or without oral administration of Liz-H. Body weight, food consumption, complete blood count, blood biochemistry, and muscle atrophy were measured. Next-generation sequencing was also performed to investigate changes to gut microbial ecology. Liz-H administration alleviated the cisplatin + docetaxel-induced weight loss, muscle atrophy, and neutropenia. Furthermore, upregulation of muscle protein degradation-related genes ( MuRF-1 and Atrogin-1 ) and decline of myogenic factors (MyoD and myogenin) after treatment of cisplatin and docetaxel were prevented by Liz-H. Cisplatin and docetaxel treatment resulted in reducing comparative abundances of Ruminococcaceae and Bacteroides , but Liz-H treatment restored these to normal levels. This study indicates that Liz-H is a good chemoprotective reagent for cisplatin + docetaxel-induced cachexia. IMPORTANCE Cachexia is a multifactorial syndrome driven by metabolic dysregulation, anorexia, systemic inflammation, and insulin resistance. Approximately 80% of patients with advanced cancer have cachexia, and cachexia is the cause of death in 30% of cancer patients. Nutritional supplementation has not been shown to reverse cachexia progression. Thus, developing strategies to prevent and/or reverse cachexia is urgent. Polysaccharide is a major biologically active compound in the fungus Ganoderma lucidum. This study is the first to report that G. lucidum polysaccharides could alleviate chemotherapy-induced cachexia via reducing expression of genes that are known to drive muscle wasting, such as MuRF-1 and Atrogin-1. These results suggest that Liz-H is an effective treatment for cisplatin + docetaxel-induced cachexia., Competing Interests: The authors declare no conflict of interest.

Cisplatin is an effective chemotherapeutic drug against tumors. Studies often report on the improvement of kidney injury by probiotics or short-chain fatty acids (SCFAs); however, the effects of SCFAs on cisplatin-induced kidney injury are rarely studied. The aim of this study is to evaluate the function of sodium acetate on preventing cisplatin-induced kidney injury. Cell viability was detected by MTT assay. SA-β-gal staining was performed to investigate premature senescence. Reactive oxygen species (ROS) production was analyzed by H 2 DCFDA staining. Propidium iodide (PI) staining was analyzed by cell cycle. Protein expression was determined by Western blot assay. Annexin Ⅴ/PI staining was used to investigate cisplatin-induced apoptosis. Tumor growth and kidney injury were evaluated in C57BL/6 mice. Sodium acetate ameliorated cisplatin-induced premature senescence and ROS production in SV40 MES-13 glomerular cells, NRK-52E renal tubular cells, and NRK-49F renal fibroblast cells. Cisplatin-induced cell cycle arrest was inhibited by sodium acetate in SV40 MES-13 and NRK-49F cells. Sodium acetate alleviated cisplatin-induced apoptosis in vivo and in vitro but not cisplatin-induced fibrosis. Our study demonstrated that sodium acetate inhibited cisplatin-induced premature senescence, cell cycle arrest, and apoptosis by attenuating ROS production. This strategy may be useful in the treatment of cisplatin-induced kidney injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cyclosporin A is an immunosuppressive drug with anti-cancer effect. Arsenic trioxide (As 2 O 3 ), a well-known cancer-inhibiting drug, induced cytotoxicity via apoptosis and autophagy. The aim of this study is to evaluate the effect of combinational treatment with cyclosporin A and arsenic trioxide on cell viability inhibition in cervical cancer cells. Using MTT assay and combination index, combinational treatment with cyclosporin A and arsenic trioxide induced a synergistic cytotoxic effect in Caski and SiHa cells. Cyclosporin A and arsenic trioxide triggered cell death via non-apoptotic pathway by using annexin V/propidium iodide (PI) assay. Cyclosporin A and arsenic trioxide combined treatment decreased mitochondrial membrane potential and increase reactive oxygen species (ROS) generation. This co-treatment increased LC3B-II expression and autophagosome formation in cervical cancer cells. This study first demonstrated that combinational treatment with cyclosporin A and As 2 O 3 trigger synergistic cytotoxic effect via autophagy in cervical cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Angiopoietin-like protein 4 (ANGPTL4) is a hypoxia-induced gene, and its high expression is associated with poor prognosis and promotion of tumour progression in several cancers. Some studies reported that ANGPTL4 is affected by epigenetic regulation. Our previous results demonstrated that ANGPTL4 is highly expressed in most lung cancer cell lines than in normal cell lines and is upregulated by HIF-1α accumulation under NiCl 2 exposure. The accurate role of ANGPTL4 and its methylation status caused by nickel in the lung carcinogenesis is not fully explored yet. In this study, we found that ANGPTL4 and HIF-1α in lung adenocarcinoma (LUAD) tissues were significantly upregulated compared with those in normal tissues in The Cancer Genome Atlas (TCGA) cohort (p < 0.001). The ANGPTL4 expression was statistically correlated to advanced stage (p = 0.019) and N value (p = 0.002). The Kaplan-Meier analysis revealed that ANGPTL4 and HIF-1α expression levels were independently associated with the 5-year survival of patients with LUAD in TCGA database and immunohistochemistry staining. In vitro experiments indicated that ANGPTL4 was upregulated by the demethylation agent. The methylation-specific PCR and bisulfite sequencing assessed the methylation status of the ANGPTL4 promoter, and results showed that NiCl 2 -treated cells had low ANGPTL4 methylation status. We further demonstrated that the DNA demethylase, TET1, was significantly increased under NiCl 2 exposure. The knockdown of TET1 expression repressed the NiCl 2 -induced ANGPTL4. We also showed that nickel-induced TET1 was stimulated by HIF-1α. Our work established ANGPTL4 as a potential oncogene that contributes to lung cancer progression and nickel-elicited carcinogenesis., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gene mutations in PIK3CA, PIK3R1, KRAS, PTEN, and PPP2R1A commonly detected in type I endometrial cancer lead to PI3K/Akt/mTOR pathway activation. Bimiralisib (PQR309), an orally bioavailable selective dual inhibitor of PI3K and mTOR, has been studied in preclinical models and clinical trials. The aim of this study is to evaluate the anticancer effect of PQR309 on endometrial cancer cells. PQR309 decreased cell viability in two-dimensional and three-dimensional cell culture models. PQR309 induced G1 cell cycle arrest and little cell death in endometrial cancer cell lines. It decreased CDK6 expression and increased p27 expression. Using the Proteome Profiler Human XL Oncology Array and Western blot assay, the dual inhibitor could inhibit the expressions of c-Myc and mtp53. KJ-Pyr-9, a c-Myc inhibitor, was used to prove the role of c-Myc in endometrial cancer survival and regulating the expression of mtp53. Knockdown of mtp53 lowered cell proliferation, Akt/mTOR pathway activity, and the expressions of c-Myc. mtp53 silence enhanced PQR309-inhibited cell viability, spheroid formation, and the expressions of p-Akt, c-Myc, and CDK6. This is the first study to reveal the novel finding of the PI3K/mTOR dual inhibitor in lowering cell viability by abolishing the PI3K/Akt/mTOR/c-Myc/mtp53 positive feedback loop in endometrial cancer cell lines.

Saracatinib is an oral Src-kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed-resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase-7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B-II in A400 cells. ATG5 silencing abolished the caspase-7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib., (© 2020 Wiley Periodicals LLC.)

Background: Adaptive drug resistance is an unfavourable prognostic factor in cancer therapy. Pemetrexed-resistant lung cancer cells possess high-metastatic ability via ERK-ZEB1 pathway-activated epithelial-mesenchymal transition. GMI is a fungal immunomodulatory protein that suppresses the survival of several cancer cells., Methods: Cell viability was analysed by MTT, clonogenic, tumour spheroid, and cancer stem cell sphere assays. Western blot assay was performed to detect the protein expression. Chemical inhibitors and ATG5 shRNA were used to inhibit autophagy. Tumour growth was investigated using xenograft mouse model., Results: GMI decreased the viability with short- and long-term effects and induced autophagy but not apoptosis in A549/A400 cells. GMI downregulated the expression levels of CD133, CD44, NANOG and OCT4. GMI induces the protein degradation of CD133 via autophagy. CD133 silencing decreased the survival and proliferation of A549/A400 cells. GMI suppressed the growth and CD133 expression of A549/A400 xenograft tumour., Conclusions: This study is the first to reveal the novel function of GMI in eliciting cytotoxic effect and inhibiting CD133 expression in pemetrexed-resistant lung cancer cells via autophagy. Our finding provides evidence that CD133 is a potential target for cancer therapy.

ABT-737, a B cell lymphoma-2 (Bcl-2) family inhibitor, activates apoptosis in cancer cells. Arsenic trioxide is an apoptosis activator that impairs cancer cell survival. The aim of this study was to evaluate the effect of a combination treatment with ABT-737 and arsenic trioxide on uterine cervical cancer cells. MTT (3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) assay revealed that ABT-737 and arsenic trioxide induced a synergistic effect on uterine cervical cancer cells. Arsenic trioxide enhanced ABT-737-induced apoptosis and caspase-7 activation and the ABT-737-mediated reduction of anti-apoptotic protein Mcl-1 in Caski cells. Western blot assay revealed that arsenic trioxide promoted the ABT-737-mediated reduction of CDK6 and thymidylate synthetase in Caski cells. Arsenic trioxide promoted ABT-737-inhibited mitochondrial membrane potential and ABT-737-inhibited ANT expression in Caski cells. However, ABT-737-elicited reactive oxygen species were not enhanced by arsenic trioxide. The combined treatment induced an anti-apoptosis autophagy in SiHa cells. This study is the first to demonstrate that a combination treatment with ABT-737 and arsenic trioxide induces a synergistic effect on uterine cervical cancer cells through apoptosis. Our findings provide new insights into uterine cervical cancer treatment., Competing Interests: There are no conflicts of interest to declare.

POU-homeodomain transcription factor POU3F2 is a critical transcription factor that participates in neuronal differentiation. However, little is known about its downstream mediators. Here genome-wide analyses of a human neuronal differentiation cell model, NT2D1, suggested neurotrophin-3 (NTF3), a key mediator of neuronal development during the early neurogenic period, as a putative regulatory target of POU3F2. Western blot, cDNA microarray, and real-time quantitative PCR analyses showed that POU3F2 and NTF3 were upregulated during neuronal differentiation. Next-generation-sequence-based POU3F2 chromatin immunoprecipitation-sequencing and genome-wide in silico prediction demonstrated that POU3F2 binds to the NTF3 promoter during neuronal differentiation. Furthermore, unidirectional deletion or mutation of the binding site of POU3F2 in the NTF3 promoter decreased promoter-driven luciferase activity, indicating that POU3F2 is a positive regulator of NTF3 promoter activity. While NTF3 knockdown resulted in decreased viability and differentiation of NT2D1 cells, and POU3F2 knockdown downregulated NTF3 expression, recombinant NTF3 significantly rescued viable neuronal cells from NTF3- or POU3F2-knockdown cell cultures. Moreover, immunostaining showed colocalization of POU3F2 and NTF3 in developing mouse neurons. Thus, our data suggest that NTF3 is a novel target gene of POU3F2 and that the POU3F2/NTF3 pathway plays a role in the process of neuronal differentiation.

β-catenin is important in development of lung cancer. In our previous study, GMI, a fungal immunomodulatory protein, inhibits lung cancer cell survival. The aim of this study is to evaluate the effect of GMI on β-catenin inhibition and apoptosis induction. GMI induced apoptosis in lung cancer cells bearing wild-type and mutated EGFR. GMI did not reduce the β-catenin mRNA expression but suppressed the protein expressions of β-catenin that resulted in the transcriptional downregulation of its target genes: survivin and cyclin-D1. The transcriptional activation activity of β-catenin was demonstrated by TOPFLASH/FOPFLASH luciferase reporter assay. Inhibition of GSK-3β and proteasome blocked the inhibiting effect of GMI on β-catenin and its target genes. β-catenin silencing increased activation of apoptosis in GMI-treated H1355 cells. This is the first study to reveal the novel function of GMI in inducing apoptosis via β-catenin inhibition. These results provide a new potential of GMI in against lung cancer., (© 2018 Wiley Periodicals, Inc.)

Autophagy is an intracellular recycling and degradation process for regulating tumor progression, survival and drug resistance. Nickel compounds have been identified as human carcinogens. However, the role of nickel-induced autophagy in lung carcinogenesis has not yet been fully elucidated. In this study, we determined that hexokinase 2 (HK2), which phosphorylates glucose and regulates autophagy, is the key mediator in nickel-induced autophagy in lung bronchial epithelial cells. We attempted to investigate the effects of the antidiabetic drug metformin on HK2 expression and lung cancer chemoprevention. Our results showed that metformin decreases nickel-induced autophagy and activation of apoptosis through inhibition of HK2 gene, protein and activity. Furthermore, we demonstrated that lipocalin 2 (LCN2), which is released by neutrophils at sites of infection and inflammation is involved in HK2-driven autophagy pathway. Knockdown of endogenous HK2 and LCN2 by shRNA reduced nickel-elicited autophagy and apoptosis, illustrating that metabolic alteration and inflammatory action are important in nickel-elicited carcinogenesis. We also determined the association between nickel-induced autophagy and apoptosis. Inhibition of nickel-induced autophagy abolished apoptotic cell death in chloroquine-treated, shLC3 Beas-2B cells and Atg5 -/- MFFs. From TGCA database and immunohistochemistry analysis, HK2 and LCN2 expression increased in lung squamous cell carcinoma and their related adjacent normal tissues. Taken together, our results demonstrated that metformin alleviates NiCl 2 -induced autophagy and apoptosis via HK2-driven LCN2 activation in human bronchial epithelial cells. This novel mechanism provides a strategy for targeting nickel-elicited lung cancer progression, as well as for preventing HK2 cumulative damage triggered by environmental carcinogens., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Hispolon, a phenolic compound isolated from Phellinus igniarius, induces apoptosis and anti-tumor effects in cancers. However, the molecular mechanism involved in hispolon-mediated tumor-suppressing activities observed in cervical cancer is poorly characterized. Here, we demonstrated that treatment with hispolon inhibited cell metastasis in two cervical cancer cell lines. In addition, the downregulation of the lysosomal protease Cathepsin S (CTSS) was critical for hispolon-mediated suppression of tumor cell metastasis in both in vitro and in vivo models. Moreover, hispolon induced autophagy, which increased LC3 conversion and acidic vesicular organelle formation. Mechanistically, hispolon inhibited the cell motility of cervical cells through the extracellular signal-regulated kinase (ERK) pathway, and blocking of the ERK pathway reversed autophagy-mediated cell motility and CTSS inhibition. Our results indicate that autophagy is essential for decreasing CTSS activity to inhibit tumor metastasis by hispolon treatment in cervical cancer; this finding provides a new perspective on molecular regulation.

Introduction: Hot flashes have been postulated to be linked to systemic inflammation. This study aimed to investigate the relationship between hot flashes, pro-inflammatory factors, and leukocytes in healthy, non-obese postmenopausal women., Participants and Design: In this cross-sectional study, a total of 202 women aged 45-60 years were stratified into one of four groups according to their hot-flash status: never experienced hot flashes (Group N), mild hot flashes (Group m), moderate hot flashes (Group M), and severe hot flashes (Group S). Variables measured in this study included clinical parameters, hot flash experience, leukocytes, and fasting plasma levels of nine circulating cytokines/chemokines measured by using multiplex assays. Multiple linear regression analysis was used to evaluate the associations of hot flashes with these pro-inflammatory factors., Settings: The study was performed in a hospital medical center., Results: The mean values of leukocyte number were not different between these four groups. The hot flash status had a positive tendency toward increased levels of circulating IL-6 (P-trend = 0.049), IL-8 (P-trend < 0.001), TNF-α (P-trend = 0.008), and MIP1β (P-trend = 0.04). Multivariate linear regression analysis revealed that hot-flash severity was significantly associated with IL-8 (P-trend < 0.001) and TNFα (P-trend = 0.007) among these nine cytokines/chemokines after adjustment for age, menopausal duration, BMI and FSH. Multivariate analysis further revealed that severe hot flashes were strongly associated with a higher IL-8 (% difference, 37.19%; 95% confidence interval, 14.98,63.69; P < 0.001) and TNFα (51.27%; 6.64,114.57; P < 0.05)., Conclusion: The present study provides evidence that hot flashes are associated with circulating IL-8 and TNF-α in healthy postmenopausal women. It suggests that hot flashes might be related to low-grade systemic inflammation.

The welding is the major working process in fitness equipment manufacturing industry, and International Agency for Research on Cancer has classified welding fumes as possibly carcinogenic to humans (Group 2B). The present study aimed to evaluate associations between the occupational exposure of metals and oxidative damage and telomere length shortening in workers involved in the manufacture of fitness equipment. The blood metal concentrations were monitored and malondialdehyde (MDA), alkaline Comet assay was determined as oxidative damage in 117 workers from two representative fitness equipment manufacturing plants. MDA levels varied according to workers' roles at the manufacturing plants, and showed a trend as cutting>painting>welding>administration workers. Welders had marginally shorter average telomere lengths than the administrative workers (p=0.058). Cr and Mn levels were significantly greater in welders than they were in administrative workers. There were significantly positive correlations between MDA and Cr and Mn levels, the major components of welding fume. However, the association would be eliminated if co-metals exposure were considered simultaneously. In future, telomere length and MDA might be potential biomarkers for predicting cardiovascular disease in co-metals exposed workers.

High thymidylate synthase (TS) level in cancer tissue is considered to result in resistance to pemetrexed therapy for advanced stages of nonsquamous non-small cell lung cancers. To further investigate the mechanism of pemetrexed resistance and potential prognostic outcomes in lung cancer, we established pemetrexed-resistant lung adenocarcinoma cell sublines from CL1 harboring a mutated TP53 gene (R248W) and A549 harboring wild-type TP53. We found the TS expression is upregulated in both pemetrexed-resistant sublines and the reduced TS level achieved through shRNA inhibition resulted in higher pemetrexed sensitivity. We also demonstrated that the acquisitions of pemetrexed resistance enhances epithelial-mesenchymal transition (EMT) in vivo with a mice animal model and in vitro with CL1 and A549 sublines, which was associated with upregulation of ZEB1 which, in turn, downregulates E-cadherin and upregulates fibronectin. When ERK1/2 phosphorylation was reduced by an inhibitor (U0126) or siRNA inhibition, both pemetrexed-resistant sublines reduced their migration and invasion abilities. Therefore, the ERK-mediated pathways induce apoptosis with pemetrexed treatment, and may in turn mediate EMT when cancer cells are resistant to pemetrexed. We further demonstrated that the growth of pemetrexed-resistant tumors could be inhibited by vinblastine in vivo and vincristine in vitro. Our data indicate that pemetrexed resistance could be relieved by non-cross-resistant chemotherapeutic drugs such as vinca alkaloids and might be independent to TP53 status. Furthermore, the phosphorylation of ERK was reduced by vincristine. This finding provides a new insight for overcoming pemetrexed resistance and metastasis by application of vinca alkaloids.

Background: Chloroquine, a lysosomal inhibitor, is used for malaria, rheumatoid arthritis, and lupus erythematosus therapy. In our previous study, FIP-gts, an immunomodulatory protein from Ganoderma tsugae, inhibited cell viability in lung cancer cells and urothelial cancer cells. Urothelial carcinoma is the most common type of bladder cancer. Cisplatin resistance is an important issue in urothelial carcinoma therapy., Purpose: The aim of this study is to investigate the effect of combination treatment with FIP-gts and chloroquine on cytotoxicity to resensitize the cisplatin-resistant cells., Methods: FIP-gts and chloroquine cytotoxicity were determined by evaluating CCK-8 assay. Cell death pathways, ROS and cell cycle arrested were analysed through flow cytometry and Western blot. ShRNA targeting to autophagy-related genes were tested to evaluate their autophagic cell death for resistant urothelial cells., Results: Using CCK-8 assay, chloroquine increased FIP-gts-induced cytotoxicity in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Caspase-3/PARP cascade and z-VAD-fmk were performed to prove that FIP-gts and chloroquine induced caspase-independent cell death. Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. To elucidate the role of autophagy in caspase-independent cell death by FIP-gts and chloroquine, LC3 shRNA were used to inhibit autophagy in N/P cells. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells. This is the first study to reveal the novel function of FIP-gts in triggering caspase-independent cell death in cisplatin-resistant urothelial cancer cells., Conclusion: Chloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation. Combination treatment with FIP-gts and chloroquine may provide a new strategy for urothelial cancer therapy., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Growth signals are directly or indirectly involved in telomerase regulation. In this study, we investigated molecular mechanisms of the effect of EGF (epidermal growth factor) on regulating hTERT (human telomerase reverse transcriptase) expression. To elucidate specific transcription factors involved in EGF-stimulated hTERT transcription in A549 and H1299 lung cancer cells, transcription factors drives hTERT promoter activity, such as Myc, Mad, and Ets-2, was evaluated on luciferase reporter assay. The upregulation of hTERT promoter by Ets-2 and Myc were abolished by Mad. Using DAPA (DNA affinity precipitation assay), Ets-2 binding to SNP (T) was stronger than Ets-2 binding to SNP (C) at -245 bp upstream of the transcription start site within the core promoter of hTERT. Ets-2 silence by siRNA decreased hTERT expression at mRNA and protein levels. The regulation of hTERT promoter by EGF/Ets-2 was diminished via the EGFR kinase signal pathway-specific inhibitors AG1478 and Iressa. Inhibitors of Erk and Akt inhibited Ets-2-activated hTERT promoter activity. These data suggested that Ets-2, a genuine cancer-specific transcription factor, is actively involved in EGFR kinase-induced hTERT overexpression pathway in lung cancer cells. Blockage of this pathway may contribute to targeted gene therapy in lung cancer.

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Cisplatin-based therapy is common in the treatment of several types of cancers, including lung cancers. In our previous study, GMI, an immunomodulatory protein cloned from Ganoderma microsporum, induced a cytotoxic effect in lung cancer cells via autophagy. The aim of this study is to examine the role of GMI in enhancing cisplatin-mediated cell death. On the basis of MTT assay and Combination Index, GMI and cisplatin cotreatment induced a synergistic cytotoxic effect. GMI and cisplatin-induced apoptosis was determined by sub-G1, nuclear condensation, and annexin-V/propidium iodide analyses. On Western blot, expressions of γH2AX and cleaved forms of PARP, caspase-3, and caspase-7 were induced by combined treatment. Akt/mTOR pathway activity, LC3-II expression, and acidic vesicular organelle development demonstrated that cisplatin does not abolish GMI-mediated autophagy. Cyto-ID Green/hoechst 33342 double staining and time-dependent experiment indicated that GMI and cisplatin-treated A549 cells simultaneously express autophagosomes and apoptotic nuclei. To elucidate the role of autophagy in inducing apoptosis by GMI and cisplatin, chemical inhibitors and LC3 shRNA were used to inhibit autophagy. The results showed that 3-methyladenine decreases, while chloroquine increases GMI and cisplatin cotreatment-induced cleavage of caspase-7 and PARP. LC3 silencing abolished activation of apoptosis in A549 cells. Caspase inhibitors and caspase-7 silencing mitigated GMI and cisplatin-elicited cell viability inhibition and apoptosis. This is the first study to reveal the novel function of GMI in potentiating cisplatin-mediated apoptosis. GMI and cisplatin induce apoptosis via autophagy/caspase-7-dependent and survivin- and ERCC1-independent pathway. GMI may be a potential cisplatin adjuvant against lung cancer.

Background and Purpose: Autophagic cell death is considered a self-destructive process that results from large amounts of autophagic flux. In our previous study, GMI, a recombinant fungal immunomodulatory protein cloned from Ganoderma microsporum, induced autophagic cell death in lung cancer cells. The aim of this study was to examine the role of autophagosome accumulation in GMI-mediated cell death., Experimental Approach: Western blot analysis, flow cytometry and confocal microscopy were used to evaluate the effects of different treatments, including silencing of ATP6V0A1 by use of short hairpin RNAi, on GMI-mediated cell death, lung cancer cell viability and autophagosome accumulation in vitro., Key Results: Lysosome inhibitors bafilomycin-A1 and chloroquine increased GMI-mediated autophagic cell death. GMI and bafilomycin-A1 co-treatment induced the accumulation of large amounts of autophagosomes, but did not significantly induce apoptosis. GMI elicited autophagy through the PKB (Akt)/mammalian target of rapamycin signalling pathway. Silencing of ATP6V0A1, one subunit of vesicular H(+)-ATPases (V-ATPases) that mediates lysosome acidification, spontaneously induced autophagosome accumulation, but did not affect lysosome acidity. GMI-mediated autophagosome accumulation and cytotoxicity was increased in shATP6V0A1 lung cancer cells. Furthermore, ATP6V0A1 silencing decreased autophagosome and lysosome fusion in GMI-treated CaLu-1/GFP-LC3 lung cancer cells., Conclusion and Implications: We demonstrated that autophagosome accumulation induces autophagic cell death in a GMI treatment model, and ATP6V0A1 plays an important role in mediating autophagosome-lysosome fusion. Our findings provide new insights into the mechanisms involved in the induction of autophagic cell death., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Endoplasmic reticulum (ER) stress is activated under severe cellular conditions. GADD153, a member of the C/EBP family, is an unfolded protein response (UPR) responsive transcription factor. Increased levels of lipocalin 2, an acute phase protein, have been found in several epithelial cancers. The aim of this study is to investigate the function of lipocalin 2 in lung cancer cells under ER stress. Treatment with thapsigargin, an ER stress activator, led to increases in cytotoxicity, ER stress, apoptosis, and lipocalin 2 expression in A549 cells. GADD153 silencing decreased lipocalin 2 expression in A549 cells. On chromatin immunoprecipitation assay, ER stress increased GADD153 DNA binding to lipocalin 2 promoter. Furthermore, silencing of lipocalin 2 mitigated ER stress-mediated apoptosis in A549 cells. Our findings demonstrated that lipocalin 2 is a new GADD153 target gene that mediates ER stress-induced apoptosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

FIP-fve is a protein that is isolated from Flammulina velutipes . Its known immunomodulatory activities are elicitation of the production of type II interferon from human peripheral mononuclear cells (hPBMCs) and hemagglutination. How the target receptors mediate activation of FIP-fve-induced immunomodulatory effects remains to be elucidated. This study postulates the three-dimensional structures to determine whether the carbohydrate binding module family 34 (CBM-34) on FIP-fve is conserved to site N of Thermoactinomyces vulgaris R-47 α-amylase I. Experimental site-directed mutagenesis data as well as ligand-specific binding competition assay are adopted to identify the key residues W24, T28, D34, T90, I91, and W111 of FIP-fve that participate in binding to polysaccharides that are linked to the membrane of immune cells. Treatments of hPBMCs with tunicamycin and deglycosylation enzymes that removed the carbohydrate moieties reduced the secretion of IFN-γ induction from hPBMCs. In conclusion, the experiments herein demonstrated the ligand-binding CBM-34 on FIP-fve and ligand-like glycoproteins on the surface of hPBMCs must be required to induce physiological immunomodulatory effects.

During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.

Curcumin is a natural compound that has been extensively observed due to its potential as an anticancer drug. Curcumin restrains cancer cell progression via telomerase activity suppression. However, the exact mechanism is still unknown. In this study, we demonstrate that the effects of curcumin on cell viability and telomerase activity can be blunted by reactive oxygen species (ROS) inhibitor N-acetyl cysteine (NAC). The ROS induced by curcumin in A549 cells was detected by flow cytometry. Using Western blot and RT-PCR, human telomerase reverse transcriptase (hTERT) decreased in the presence of curcumin. Sp1 is one of the important transcription factors in hTERT expression. Our data showed that curcumin decreases the expression of Sp1 through proteasome pathway. In addition, NAC blunted the Sp1 reduction and hTERT downregulation by curcumin. Further, reporter assay and DNA affinity precipitation assay confirmed the influence of curcumin on Sp1 in hTERT regulation. This is the first study to demonstrate that curcumin induces ROS production resulting in Sp1 binding activity inhibition and hTERT downregulation., (Copyright 2010 Elsevier B.V. All rights reserved.)